In particular, in the field of regenerative medicine using tissue engineering, the use of stem cell technology is becoming a new territory for intractable disease treatment. Accordingly, stem cell research is getting more attention. Stem cells are recognized as a problem solver not only for curing disease but also for repairing damaged tissues.
Stem cell differentiates into particular cells under an appropriate condition, by having replicative ability when it is still undifferentiated. Stem cell can be classified into Embryonic Stem Cell and Adult Stem Cell, depending on its origin. Human embryonic stem cell is acquired from an embryo that can grow to a human being. Therefore, it has an excellent cellular proliferation and differentiation ability, but is tied with life ethics. Adult stem cell is limited with differentiation ability, compared with embryonic stem cell. However, it is developed into a stem cell by taking from cells inhuman organs such as marrow, blood, brain, skin, etc. Therefore, it has less life ethics issues.
Mesenchymal Stem Cell is separated from adult marrow at first (Y. Jiang et al., Nature, 418:41, 2002). After that, it is confirmed that there found mesenchymal stem cell just like marrow, from skin, blood, muscle, brain tissues (J. G. Toma et. al., nat. Cell Biol., 3:778, 2001:M, Sampaloesi et al., Science, 301:487, 2003: Y. Jiang et al., Hematol., 30:896, 2002). Also, it is found that Adipose-derived Stem Cell that is acquired from fatty cell has the same differentiation potency like marrow (B. Cousin et al., BBRC., 301:1016, 2003).
The method of separating mesenchymal stem cell from human marrow and the method of separating mesenchymal stem cell from fatty cells are explained in Pittenger et al., (Science 284:143, 1997) and van et al., (J. Clin, Invest., 58:699, 1976). These literatures employ alpha-MEM, DMEM medium and 10-20% of small fetal serum for culturing cells.
However, there rarely exists any mesenchymal stem cell in adult cell (such as marrow, fatty tissue, etc.). In addition, these cells have low proliferation rate when they are undifferentiated. If undifferentiated, it is difficult to keep it for a long term. Therefore, if there is no screened medium, it is known to be difficult to multiply/culture/preserve in vitro.
The composition of mammal's cell culture medium consists of about 50 different kinds. They are classified into the followings: 1) a part that is used for cellular biosynthesis, 2) a part that is used for biological energy metabolism, 3) a part that is used either to act as a catalyst for various metabolisms or to adjust intracellular physiological phenomena. That is, the medium that is used for cellular culturing consists of composition of isotonic solution and buffer solution, nutrients (that include sources of energy such as amino acid, vitamins, inorganic base, etc.), and various types of supplements.
Depending on the cellular type, it provides hormones, growth factor, fat, vitamins, and suppresses the activation of protein breakdown enzyme, by providing 5-20% of blood serum. Then it promotes the growth and revitalization of mammal cells, by acting as a pH adjusting buffer. The composition of medium to culture mammal cells varies depending not only on concentration but also on the type of medium. With this method, M119 medium is made and therefore primary chick is cultured (Morgan, et al., 1950), based on body fluid composition in 1950's.
For the type of culturing medium, there are media that are used in common and simple for culturing cells, as shown in Minimal essential medium (MEM) (Eagle, 1955) and Dulbecco's modification of Eagle's medium (DMEM) (Dulbecco, et al., 1959). Just like Isocove's modification of DMEM (IMDM), Ham's F-12 (Ham, 1965) and Connaught Medical Research Laboratories (CMRL)-1666, it is possible to classify into media with more being complex and with more various components.
The growth curve of mammal cells has the lag phase of 2-3 days. The concentration of living cells significantly decreases from the end of lapse period due to the accumulation of lactate (the end product of glucose) and ammonium (the end product of glutamine metabolism). The main metabolic pathway of mammal cell is used as the main source of carbon and energy of glucose and glutamine. Once glucose is metabolized by mammal cell, it becomes Pyrubic Acid through glycosis. Also, it synthesizes hexane by making pentose by Pentose phosphate pathway. Pyrubic acid that is made through glycosis is disintegrated into CO2, H2O, lactate or fatty acid with TCA circuit among those carbon source that are used in cellular metabolism for mammals, apart of Glutamin becomes Glutamate during metabolism process. Glutamate enters into TCA circuit, and makes a Carbon Skeleton for the synthesis of other amino acids. Main istes of mammals are lactate and ammonia. However, the discharge of alanine is also significant. Lactate and ammonia change the pH of inside of cells and lysosome, such that they can be poisonous to cells. Likewise, the type of medium for culturing should be different, because they are different in physiological mechanism and nutrient requirement depending on the type of cultured cells.
Therefore, in order to proliferate/culture adult cell derived (marrow, fatty cells, etc.) undifferentiated mesenchymal stem cell, the condition of medium should vary depending on the growth condition of undifferentiated mesenchymal stem cell.
There have been a number of researches conducted on culturing medium in vitro that multiplies/cultures undifferentiated mesenchymal stem cell.
Patent Literature 1 (a method of mass production of growth factor using mesenchymal stem cell) is based on DMEM. Adding Ham's F-12 will accelerate the differentiation of growth factor from mesenchymal stem cell. Accordingly, it describes about serum-free medium that manipulates to synthesize significant amount of human growth factor. However, this is to mass produce basic fibroblast growth factor, venular endothelial cell growth factor or human transforming growth factor-beta, rather than the proliferation of mesenchymal stem cell in a basic medium that is mixed with DMEM and Ham's F-12.
Patent Literature 2 (medium composition that is required for cord blood-derived mesenchymal stem cell that includes soybean protein hydrolysate) is a technology to add soybean protein hydrolysate to hypoglycemosis DMEM (including fetal bovine serum), in order to reduce the amount of fetal bovine serum.
Patent Literature 3 (a method of manufacturing papilla tissue using mesenchymal stem cell) and Patent Literature 4 (a method of manufacturing papilla tissue using mesenchymal stem cell) are technologies to induce differentiation into papilla tissues, after culturing mesenchymal cells in DMEM medium, DMEM/F-12, F-12, McCoy's 5A, RPMI 1640 medium, Williams's medium E, or IMDM (Iscove's Modified Dulbecco's Modification). This relates to medium added with hydrocortisone, insulin, Transferrin and Sodium selenite in commercialized basic cell culturing medium.
Patent Literature 5 (mesenchymal stem cell culturing medium & a method of culturing mesenchymal stem cell using it) is a technology to culture by adding insulin, hydrocortisone, EGF, LIF, GM-CSF, etc. based on mixed medium (that is added with nutrient mixture) in commercialized medium. This is a technology to mix nutrient mixture in already-commercialized culturing medium.
Patent Literature 6 (adipose pluripotent stem cell & a cellular therapy product that contains it) is based on DMEM medium. It is about technology to multiply mesenchymal stem cell by adding Keratinocyte-SFM medium (that is also added with NAC, rEGF, BPE, insulin, etc.). Its basic cell culturing medium is DMEM medium.
Patent Literature 7 (culture of adult stem cell or its fraction, composition of pharmaceutical treatment and cancer treatment) and Patent Literature 8 (separated pluripotency adult stem cell and its separation/culturing) are technologies to culture mesenchymal stem cell by using DMEM/Ham's F-12 mixed medium, DMEM medium and DMEM/F-12.
These conventional technologies are limited to those that culture previously commercialized medium by adding additives such as growth factor.
However, our patent (Patent Literature 9) (a method of special culturing of cartilage cells for early culturing of cartilage cells) is the one to develop culture medium without adding additives such as growth factor, etc. By using early-cultured medium (ABM-C) of Patent Literature 9, the mesenchymal stem cell (one that is separated from adult cells such as marrow and fat) is cultured. However, there found morphological anomaly during cell culturing as shown in FIG. 13. Also, without adhesiveness, it floats on cell culturing medium. Therefore, mesenchymal stem cell does not proliferate, the one that multiplies being attached to in vitro.